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Research has shown that autologous progenitor-like cardiac spheroids, when delivered to an infarcted heart, are able to restore mechanical function. These spheroids are made by isolating and expanding autologous cardiac progenitor cells. Though these results are promising, the process for creating cardiac spheroids is inefficient and time consuming, requiring a large amount of cardiac tissue. For every 10,000 cardiac myocytes in the heart there is only one cardiac progenitor cell; requiring a large amount of initial tissue. This clinical limitation could be overcome if cardiac myocytes, which are more abundant than cardiac progenitor cells, could be used to make cardiac spheroids. Research has shown that mesenchymal stem cells when co-cultured with adult cardiac myocytes cause the cardiac myocytes to behave like a progenitor cell. We found that, when co-cultured with mesenchymal stem cells, cardiac mycoytes could be made to form cardiac spheroid bodies. This was done by isolating adult myocytes from rat hearts and co-culturing them with human mesenchymal stem cells. After two weeks, cultures were observed to form spheroid bodies and the number of spheroids formed were compared to a pure myocyte control. Cardiac specific staining confirmed that the spheroids were made from the myocytes. It was also found that the mesenchymal stem cells, when co-cultured in the same well with the myocytes, form significantly more spheroids than myocytes treated with stem cell conditioned media. Further, no other cell type present in the co-cultures are able to create spheroid bodies when co-cultured with mycoytes or stem cells. The ability to create cardiac spheroid like bodies from adult myocytes offers a way to overcome the limitations of the time needed and the large quantity of autologous cardiac tissue required to currently make these types of bodies.

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This study investigated the effect of the mouse estrous cycle on myocyte contractile function. Female mice displayed irregular estrous cycles unless induced to cycle though exposure to bedding collected from cages housing male mice. Fractional shortening and Ca2+ transient amplitudes were significantly larger in myocytes isolated from mice in estrus. The effect of the estrous cycle was preserved even when cells were paced at a more physiological frequency and in the presence of ?-adrenergic stimulation. Myofilament Ca2+ sensitivity was also modified by the estrous cycle, as myofilaments isolated from the hearts of mice in estrus were least sensitive to Ca2+. However, acute application of either 17?-estradiol or the G protein-coupled estrogen receptor (GPER) agonist, G-1, had no effect on contractions or Ca2+ transients, regardless of the estrous stage. Thus, physiological fluctuations in sex hormone levels modify myocyte contractions, Ca2+ release, and myofilament Ca2+ sensitivity.

In heart, Ca2+ plays an important role in maintenance of normal cardiac functions. Regulation of Ca2+ is mainly through L-type Ca2+ channel (LTCC), Ryanodine receptor (RyR) and sarcoplasmic reticulum calcium ATPase pump (SERCA) on sarcoplasmic reticulum (SR), Na+-Ca2+ exchanger (NCX), plasma membrane Ca2+ ATPase (PMCA). It has been well-accepted that PMCA plays a minor contribution to elevation of Ca2+ compared to SERCA and NCX and in regulation of cytosolic Ca2+ homeostasis. There are four isoforms of PMCA, PMCA1-4, and PMCA1 is a house-keeping gene, and abundantly distributed in heart. However, the role of PMCA1 in the murine heart has not been fully explored. With a cardiac specific knockout mouse model, the electrophysiological characteristics of PMCA1 in murine hearts, particularly in atria under normal physiological and stress conditions ([Ca2+]o overload and pacing conditions) are investigated. Firstly the complete deletion of PMCA1 in the atria in PMCA1cko mice was confirmed by Western blotting and immunostaining, also the membrane localisation of PMCA1 in the atria in PMCA1loxP/loxP mice was demonstrated. Then the phenotypes of ex vivo whole hearts between PMCA1loxP/loxP and PMCA1cko mice under physiological conditions and [Ca2+]o overload condition and with different frequencies by programmed electrical stimulation (PES) were explored. Further more, the Ca2+ handling process in single atrial myocytes between the PMCA1 deletion mice and control mice under normal physiological conditions and [Ca2+]o overload condition and stimulation with different frequencies was investigated. Finally the Ca2+ handling process in single ventricular myocytes between the PMCA1 deletion mice and control mice under normal physiological condition was investigated. At the whole heart level, the PMCA1cko hearts became more susceptible to arrhythmias with PES under physiological conditions compared with the PMCA1loxP/loxP hearts, and such arrhythmic events occurred more often and had longer pacing durations under Ca2+ overload conditions and higher frequency of pacing. At the single cellular level, the NCX current decay was significantly prolonged in PMCA1cko atrial myocytes under physiological conditions. This was further increased under Ca2+ overload conditions. With frequency-dependent stimulation, the PMCA1cko atrial myocytes showed few EAD- or DAD-type APs under physiological conditions in contrast to PMCA1loxP/loxP atrial myocytes that showed no arrhythmic events. The occurrence increased significantly under Ca2+ overload condition and/or at higher frequency of stimulation. Similar findings were observed in isolated ventricular myocytes. To conclude, the role of PMCA1 in maintaining Ca2+ homeostasis and electrical function in atrial myocytes under physiological conditions is minor. ii) PMCA1 has a critical role in maintaining Ca2+ homeostasis and electrical function in the atrium under stress conditions. This is particularly important during fast efflux of Ca2+ which is required under stress conditions.

Although adenosine receptors have been implicated in the induction of preconditioning in a variety of experimental models, there is controversy concerning the specific adenosine receptor subtypes mediating this effect. Concentration-protection relationships for adenosine and adenosine agonists in rabbit cardiomyocytes were used to characterize the role of adenosine receptor subtypes in preconditioning. Isolated cells were ischemically preconditioned or pre-incubated for 10 min with increasing concentrations of adenosine, CCPA (2-chloro-N6-cyclopentyladenosine) APNEA (N6-2-(4-aminophenyl)ethyladenosine), or BNECA (N6-benzyl-5'-N-ethyl-carboxamidoadenosine) in the presence or absence of 1 or 10 μM of the selective A1-adenosine antagonist DPCPX (8-Cyclopentyl-1,3-dipropylxanthine). Following a 30-min post-incubation period, cells were pelleted, layered with oil and ischemically incubated for 180 min. Injury was assessed by osmotic swelling and trypan blue exclusion of sequential samples, and determination of the areas beneath the mortality curves. Adenosine produced a broad concentration-protection curve which was displaced to the right by DPCPX. The curve for A1-selective agonist CCPA was biphasic, with an initial response below 1 nM and a second above 1 μM. DPCPX abolished the early response leaving a steep monophasic curve between 0.1 and 10 μM CCPA. The APNEA curve appeared monophasic, the major slope occurring between 1-100 nM; DPCPX (1 μM) shifted the concentration-response curve ≃ 30-fold and decreased the slope. Adenosine receptor agonist BNECA produced preconditioning characterized by a shallow monophasic concentration-protection curve with a maximal effect of 49% and an EC50 of ≃ 5 nM; DPCPX shifted the BNECA concentration-protection relationship ≃ 40-fold with only a modest increase in slope. Analysis of the data suggests that induction of preconditioning results from interaction of agonists with the A1 receptor and a second adenosine receptor having properties consistent with the A3 receptor. Adenosine, CCPA, APNEA, BNECA and DPCPX each appear to be selective for the A1 adenosine receptor subtype in isolated rabbit cardiomyocytes.